Processes for the purification of waste effluents

ABSTRACT

A PROCESS IS DISCLOSED FOR REMOVING PROTEIN FROM WASTE EFFLUENTS CONTAINING SAME USING ION EXCHANGE METHODS THE ION EXCHANGE RESIN IS A CROSS LINKED REGENERATED CELLULOSE MATRIX MODIFIED BY THE INTRODUCTION OF GROUPS CAPABLE OF CATION EXCHANGE OR GROUPS CAPABLE OF ANION EXCHANGE.

p 0. 1 14 M. GRm-r 3,835,041

PROCESSES FOR THE PURIFICATION Filed July 3. 1972 3 Shoeta-Sheat 2United States Patent 3,835,041 PROCESSES FOR THE PURIFICATION OF WASTEEFFLUENTS Roy Arthur Grant, Poole, England, assignor to Tasman VaccineLaboratory Limited, Upper Hutt, New Zealand Continuation-impart ofapplication Ser. No. 841,430, July 14, 1969, now Patent No. 3,697,419.This application July 3, 1972, Ser. No. 268,299 The portion of the termof the patent subsequent to Mar. 30, 1988, has been disclaimed Int. Cl.B01d 15/04 U.S. Cl. 210-27 2 Claims ABSTRACT OF THE DISCLOSURE A processis disclosed for removing protein from waste efiiuents containing sameusing ion exchange methods. The ion exchange resin is a cross linkedregenerated cellulose matrix modified by the introduction of groupscapable of cation exchange or groups capable of anion exchange.

This application is a continuation-in-part of my copending applicationSer. No. 841,430, filed July 14, 1969, now Pat. No. 3,697,419.

This invention relates to processes for the purification of wasteefiluent containing protein.

Meat works, abattoirs, fish canneries, dairies, glue works and the likeindustrial concerns handling animal products produce large volumes ofeffluent. Such efiiuent may vary in composition, but a typical efiiuentfrom an abattoir results from the use of large quantities of water forthe washing of slaughterhouse equipment and carcasses. It thus containsappreciable amounts of blood proteins, soluble protein from muscle andother tissues, and sus pended or colloidal protein material, togetherwith a certain amount of particulate tissue fragments and fat. Al thoughthe concentration of protein in the effluent is usually quite low (aboutA: lb. per 100 gals.) the solution is capable of nourishing abundantundesirable organisms. It has been estimated that in a typicalslaughterhouse the material lost in the efiiuent constitutes about 5% ofthe weight of slaughtered animals, the remaining 95% of the weight beingutilised in one way or another. Another source of protein containingeffluents are natural rubber latex processing factories.

Some factories producing protein-containing eflluents may be so sitedthat the efiluent can be discharged directly into a river or into thesea without treatment. However, where this is not possible, it is usualto treat the efiluent to reduce the concentration of proteins and othernutrients before discharging the effluent into a waterway in order toprevent the pollution effects of the undesirable organisms that may growon the untreated efiluent as Well as to prevent the stench caused by thedecomposing animal matter.

At present, effiuents such as those from meat works are usually treatedby biological treatment in holding tanks. Such biological treatmentinvolves both aerobic and anaerobic degradation of the organic materialin the effluent. This has the double disadvantage that a large area ofground is required for the equipment and that the process destroys thepotentially valuable proteins. Furthermore the expense of providingsuitable holding tanks is considerable.

Where it is desired to discharge the effluent directly into a municipalsewer the authorities responsible for treating the sewage may levy aconsiderable charge on the meat works for disposing of the effluentbecause of the capital expenditure involved in providing suitableholding tanks and the running costs of operating the process.

It has been proposed (U.S. Pat. No. 2,446,913Erlich to pass the slop orthin stillage remaining after the alcohol has been distilled off from afermented mixture of wheat and 1.0% barley malt through beds of an ionexchange resin after first having centrifuged the slop or thin stillage.The slop or thin stillage, before passage through the bedsof ionexchange resin, is said to contain proteinaceous compounds in additionto salts and a nitrogen free extract. The proportion of proteinaceouscompounds is estimated by performing a nitrogen analysis and multiplyingthe result by 6.25. However, there is no unambiguous proof that theproteinaceous compounds are true proteins. The proteinaceouscompounds-containing solution is passed first through a cation absorbingtype (C) synthetic phenol-formaldehyde resin and then through an anionabsorbing type (A) synthetic amine-formaldehyde resin. O11 regeneratingthe resins and combining the regenerants a solid is obtained which isstated to contain 29% proteinaceous compounds in addition to variousinorganic salts. However there is no proof given that the proteinaceouscompounds are true protein materials, the figure of 29% being merelybased upon a nitrogen analysis the result of which is multiplied by6.25. Even ammonium salts can be classed as proteinaceous compound onthis basis.

It is also known to extract proteins on a laboratory scale from aqueoussolutions thereof by passage through a column of diethylaminoethylcellulose (DEAE-cellulose). An example of the use of DEAE-cellulose forextracting protein from solution i described in U.S. Pat. No. 3,409,605(Florini). However, although DEAE-cellulose is successful on alaboratory scale where very low flow rates can be tolerated, it isimpracticable to use DEAE-cellulose resins for industrial purificationof raw protein-containing eflluents because the D'EAE-cellulose resinsvery quickly become blocked and the beds of the DEAE-cellulose becomechoked so that the flow rate drops to an unacceptably low level.

It is an object of the present invention to provide a process forpurifying a waste efiluent by removal of proteins therefrom which can bepracticed using equipment of low capital cost and occupying only a smallground area.

It is a further object of the present invention to provide an ionexchange process for purifying raw protein-containing effluents toremove protein therefrom.

It is yet another object of the present invention to provide a processfor purifying protein-containing efiluents by removing protein therefromwithout destroying the protein.

It is a still further object of the present invention to provide aprocess for purifying efiluents from meat works, slaughterhouses, fishcanneries, and the like by removing protein from such efiluents inrecoverable form.

Essentially, the present invention is directed to a process for thepurification of a waste effiuent containing protein comprising the stepsof:

(a) passing the said effluent through a bed of a granular ion exchangematerial in the form of a cross-linked regenerated cellulose modified bythe introduction of cationic or anionic exchange groups to removeprotein from said effluent,

(b) recovering essentially protein-free effluent, and

(c) subsequently regenerating the ion exchange material for use in afurther cycle.

The ion exchange resin for use in the process of the present inventionis a cross-linked regenerated cellulose modfiied by the introduction ofcationic or anionic exchange groups. Such resins are described in U.S.Pat. No. 3,573,277. The preferred form of regenerated cellulose used inmaking the ion exchange resin is viscose.

The ion exchange material may contain groups capable of anion exchangeor groups capable of cation exchange. Typical groups capable of cationexchange which may be present in the ion exchange material includecarboxylic acid groups, sulfonic acid groups and phosphate groups andsalts thereof. Typical groups capable of anion exchange which may bepresent in the ion exchange material include amino, optionallysubstituted monoalkyl amino, optionally substituted dialkylamino,optionally substituted arylamino, guanidino and quaternary ammoniumgroups. The ion exchange groups may be bound to the regeneratedcellulose chains of the cross-linked regenerated cellulose matrixthrough brodge members such as alkylene (eg. methylene, ethylene, 1,2-or 1,3- propylene, or 1,4-butylene), alkylene-oxy-alkylene (e.g.ethylene-oxy-ethylene), hydroxyalkylene and the like.

Cross-linking of the regenerated cellulose may be carried out usingessentially any at least bifunctional compound capable of reacting withtwo hydroxyl groups to form covalent bonds under suitable conditions.Suitable cross-linking agents include aldehydes, diisocyanates,dicarboxylic acids and anhydrides, and compounds containing at least tworeactive groups selected from the group consisting of halogen atoms andepoxide groups, for example reactive dihalogenated compounds, reactivediepoxides, diisocyanates, and compounds containing a reactive halogenatom and a reactive epoxide group. Thus typical cross-linking agentsinclude formaldehyde, glyoxal, glutaraldehyde, acrolein, epichlorhydrin,dichlorhydrin, butanediol bisepoxypropyl ether, bisepoxypropyl ether,ethylene glycol bisepoxypropyl ether, phosgene, oxalyl chloride, adipoylchloride, 2,4,6-trichlorotriazine,2-methy1-5-bromomethyl-4,6-dichloropyrimidine, 2. phenylamino-4,6dichlorotriazine, p dibromomethylbenzene, a,a'-dichloroacetone, phthalicanhydride, maleic anhydride, 1,4-dichlorobutane, 1,6-dibromohexane,toluene diisocyanate, p,p diisocyanatodiphenylmethane,naphthalene-1,5-diisocyanate, metaphenylene diisocyanate, bitolylenediisocyanate and the like. Cross-linking may also be achievedphysically, for example by exposure of the regenerated cellulose to highintensity ionising radiation (such as that aiforded by ultravioletlight, gamma rays or electron beams) in the presence of water andsensitizing chemicals. In a preferred ion exchange material thecross-linking is provided by aldehyde residues, such as formaldehyderesidues, produced by treatment of the re generated cellulose with analdehyde under acid conditions. In other especially preferred materialsthe crosslinking is achieved by treatment with epichlorhydrin ordichlorhydrin under basic conditions. Preferably the degree ofcross-linking in the ion exchange material varies between 1 and percent,especially from I to 2 percent. The degree of cross-linking is expressedin terms of the ratio of the weight of cross-linking agent reacting witha given dry weight of the regenerated cellulose. This is readilymeasured by noting the weight of cross-linking agent that is added forcross-linking and subtracting the weight of cross-linking agent thatremains (if any) at the end of the cross-linking reaction.

The ion exchange groups are introduced into the regenerated celluloseeither simultaneously with or after cross-linking. To introduce groupscapable of cation exchange there may be used, for example, chloro orbromo substituted carboxylic and sulfonic acids and salts thereof, forexample chloracetic acid, bromacetic acid, chloropropionic acid,chloromethane sulfonic acid, bromoethanesulfonic acid,chloroethanesulfonic acid and salts, preferably alkali metal andalkaline earth metal salts thereof, and the like as well as1,3-propanesultone. As examples of compounds suitable for introducinggroups capable of anion exchange there may be mentioned chloro and bromosubstituted tertiary amines and salts thereof, such as,B-chloroethyldiethylamine, fl-bromoethyldiethylamine,fl-chloroethyldimethylamine, fl-bromoethyldimethylamine,chloromethyldimethylamine, bromomethyldimethylamine,chloromethyldiethylamine, bromomethyldiethylamine,B-chloroethyldifi-hydroxyethyl amine, B- bromoethyldifl-hydroxyethylamine, chloromethyldi-( B- hydroxyethyl) amine, bromomethyldi(ti-hydroxyethyhamine, N chloroethylrnorpholine,N-fi-bromoethylmorpholine, N-bromornethylmorpholine,N-chloromethylmorpholine, and their salts, for example thehydrochlorides and hydrobromides thereof, and the like. Quaternaryammonium groups may be introduced by the use of quaternised forms of theabove-mentioned amines, for example the quaternary salts obtained bytreatment of such amines with methyliodide, methylsulfate, ethylsulfate,benzyl chloride or the like. Quaternation may take place before or afterreaction with the regenerated cellulose. It is also possible tointroduce groups capable of anion exchange by reaction withepichlorhydrin and amines such as ethanolamine, isopropanolamine,diethanolamine, diisopropanolamine, triethanolamine,triiosopropanolamine, dimethylaminoethanol, diethylaminoethanol,diisopropylaminoethanol and the like. In this case the epichlorhydriu isintended to attach the amino groups to the regenerated cellulose chains,although if an already cross-linked regenerated cellulose is reactedwith a mixture of epichlorhydrin and one of the specified amines theepichlorhydrin may incidentally introduce further cross-linking residuesinto the resin matrix.

Methods for determining the ion exchange capacity of an ion exchangematerial, that is to say its ability .to exchange small ions such ascalcium or chloride, are well known. The ion exchange capacity isexpressed in milliequivalents exchangeable ions per gram of the dry ionexchange material. Various standard methods for determining the ionexchange capacity are described, for example, in Kunin Ion ExchangeResins, New York, 1958 (see pages 343345). In the process of the presentinvention it is preferred to use ion exchange materials having an ionexchange capacity for small ions of between about 0.5 and about 2milliequivalents per gram, more especially about 1 milliequivalent pergram.

In the process of the present invention, the waste effluent may, ifdesired, be submitted to a pro-treatment step to remove at least some ofthe protein and/or fat in the efiluent. In one form or pro-treatment theefiluent is collected in a holding tank and air is blown into thecollected efiluent thereby subjecting it to air flotation, Uponstanding, a scum which mainly consists of protein and fat, forms on thesurface of the efiluent and can be scraped olf.

Other methods of pre-treatment include filtration to remove suspendedmatter.

By the term protein there is meant a material which may contain someprotein breakdown products (i.e. amino acids and/or polypeptides) sincethese will almost inevitably be present in protein containing efiluents,but which is predominantly in the form of protein macromolecules. Ingeneral, solutions of macromolecular protein material can be dialysed(i.e. the protein material does not penetrate a semi-permeablemembrane), exhibit absorbance in their U.V. spectra (ultravioletspectra) at about 280;; (due to the presence of tyrosine residues) andgive a white precipitate on addition of phosphotungstic acid solution.

The ion exchange material used in the process of the invention has onlya limited ion exchange capacity. A bed of the ion exchange material offinite size can only pick up a finite amount of protein from solutionsthereof. Thus it is periodically necessary to stop the passage ofeffiuent through the bed of ion exchange material and to regenerate thebed of ion exchange material for use in a further cycle. Although it ispossible to use a single bed of ion exchange material and toperiodically interrupt the flow of efiluent whilst the bed isregenerated, it is preferred to operate the process using a plurality ofbeds of ion exchange material, the flow of effluent being periodicallyswitched from one bed to another so that regeneration of the beds can beaccomplished in turn without interrupting the flow of effiuent.

Although for many purposes it is suflicient to pass the effluent througha single bed of resin, in some cases it is desirable to pass theefiluent through several beds in series. For example the efiluent can bepassed through one or more beds of resin having groups capable of cationexchange and then through one or more beds of resin having groupscapable of anion exchange, or vice versa.

Regeneration of the bed or beds of ion exchange resin can beaccomplished by use of a suitable regenerant solution. Typical solutionsthat may be used to regenerate the ion exchange material are aqueoussolution of inorganic salts such as sodium chloride, potassium chloride,sodium sulfate, potassium sulfate, sodium bromide, sodium acidphosphate, disodium phosphate, trisodium phosphate, and the like andmixtures thereof. To such inorganic salts solutions may be added acidsor bases. Preferred are strong mineral acids such as hydrochloric acid,sulfuric acid and the like, and strong alkalis such as sodium hydroxide,potassium hydroxide, sodium carbonate, potassium carbonate and the like.The regenerant solution may contain at least 0.1% but preferably notmore than by weight of acid or alkali and even more preferably not morethan 5% by weight. The concentration of salt may vary, for example, from0.1% by weight up to 10% or more by weight, but is preferably in therange of 1% to 5% by weight of the regenerant solution. Typicalregenerant solutions are 3% by weight brine solutions containing either1% sulfuric acid or 1% sodium hydroxide. Although in most cases it isunimportant whether a neutral, acid or alkaline regenerant solution isused, some proteins are sensitive in particular to acid and may beprecipitated by acid. In such a case use of an acid regenerant solutioncould lead to formation of a precipitate of protein on the ion exchangematerial. It is therefore recommended to test a portion of an eflluenton a laboratory scale to make sure that all the protein absorbed canalso be desorbed before embarking on large scale regeneration of theexhausted resin. In general it is, however, preferred to use an acidicregenerant if the resin contains grouns capable of cation exchange, suchas carboxylic or sulfonic acid groups, and to use an alkaline regenerantif the resin contains groups capable of anion exchange.

In a preferred process the bed (or each bed if more than one is used) ofion exchange material is periodically backwashed, that is to say theregenerated bed is washed by passing water through the bed in theopposite direction to the direction in which the efiluent is passed.

After regeneration more efiluent can be passed through the bed ofregenerated ion exchange material and the cycle repeated.

The regenerant solution after passage through the spent bed of ionexchange material can be further treated as described in my co-pendingApplication Serial No. 268,- 282 filed simultaneously herewith in orderto recover protein. (Co-pending Patent Application Serial No. 268.282 isa continuation-in-part of my Application Serial No. 161,110 filed 9thJuly 1971, now abandoned, which was divided out of my co-pending PatentApplication Serial No. 841,430 filed 14th July 1969.)

Typically the process of the invention can be applied to efiluents suchas slaughterboard washings, paunch washings, skin washings, dairyeffluents, glue works eflluents, natural rubber latex factory eflluents,fish cannery effluents, and the like.

The method of pretreatment, if one is used, is largely determined by thenature of the effluent and such factors as the source of the protein orproteins in it, the concentration of protein in the efiiuent and thepresence of other constituents such as fat. The choice of ion exchangematerial, i.e. whether it contains groups capable of cation exchange oranion exchange, is largely determined by the source of the protein orproteins. In some cases it is more satisfactory to use, for example aDEAE-form (diethylaminoethyl form) of cross-linked regenerated celluloseand in others, for example, a CM-form (carboxymethyl form). In yet otherinstances it may be necessary to use first a DEAE-form and then aCM-form, or vice versa, in order to obtain the desired purification ofthe effluent.

The essentially protein-free solution recovered after passage of theeffluent through the ion exchange material has a low COD. and B.O.D.value and may be discharged to waste or it may be reused for suchpurposes as washing of equipment, or washing down the slaughterboard.However if desired it may be further purified by passage through ascavenging bed of a resin in fibrous or sponge form such as, forexample, diethylaminoethyl cellulose (DEAE-cellulose) or carboxymethylcellulose. Alternatively, or in addition, the resulting essentiallyprotein-free efiluent may be passed through a percolating filter or bonechar, activated carbon or coke filter.

Although gradient elution can be used for regeneration of an exhaustedbed of resin (i.e. one that has taken up protein from solution), it isusually preferable to elfect regeneration by stepwise elution in orderto keep the volume of protein-containing regenerant solution small. Inthis way substantially all the protein adsorbed by the resin can berecovered as a relatively concentrated solution of protein from whichprotein can be relatively simply recovered. Using a suitable regenerantsolution (e.g. 3% NaCl+l% NaOH or 1% H SO the adsorbed protein cangenerally be recovered in a small volume, e.g. about 1 bed volume, ofregenerant. In a preferred process the effluent level is adjusted sothat the bed of resin is just covered by liquid and therefore contains 1bed volume of liquid. Regenerant solution is then slowly added to thetop of the bed of resin. The U.V. absorbance of the eluate at 280 mrises from zero very sharply just after a volume of regenerant has beenadded corresponding to the bed volume. At this point the flow solutionthrough the bed can be interrupted for a period, for example for about 1hour, in order to allow the bed of resin to equilibratc with theregenerant solution. The regenerant solution can then be run out of thebed, its place being taken by a slow stream of pure water. In this waysubstantially all the adsorbed protein is desorbed in about 1 bed volumeof regenerant. Alternatively the flow of regenerant can be continuousthrough the bed of resin the progress of regeneration being monitored bymeasuring the U.V. absorbance of the eluate at 280 m Regeneration iscomplete when the absorbance returns substantially to zero.

To recover protein from the regenerant solution from the resin bed avariety of techniques may be used. In one preferred process the pH ofthe regenerant from the resin bed is adjusted to 4.5 to 5.0. Ideally oneshould ad ust the pH to correspond with the isoelectric point of theprotein or proteins in the efiluent but, since the effluent usuallycontains a variety of proteins, a compromise value for the pH may haveto be adopted. For efiluents from animal sources it is usuallysatisfactory to adjust the pH at this stage to 4.5 to 5.0. At this pointa precipitate of protein may appear. In order to coagulate the pre-. cpitated protein and to facilitate separation of the precipitated proteinfrom the supernatant liquid the solution may be heated to a temperatureof, for example, C. to C. or higher (if superatmospheric pressure isused). Heating may be accomplished externally but a convenient method ofheating the solution is to blow steam through it. The coagulated proteincan be separated by filtration or centrifugation.

In an alternative technique, after adjustment of the pH of theregenerant solution from the bed of resin to 4.5 to 5.0, a flocculantmay be added in order to precipitate the protein. If the protein is tobe used for nutritional purposes, e.g. for feeding poultry, a non-toxicflocculant should be used, for example sodium hexametaphosphate,

ligninsulphonic acid or a mixture of so-called Triple Super Phosphateand alkali. If necessary the solution can be heated as before in orderto ensure coagulation, followed by filtration or centrifugation.

The ion exchange resin is preferably in granular or particulate formhaving a size of preferably 20 to 200 mesh, or even more preferably 50to 100 mesh (British Standard 410/62).

Comparative Experiments A series of experiments was made to test theability of various ion exchange resins to take up protein from solution.In each case a bed of resin of approximately 60' ml. bed volume was madeup by slurrying a suitable amount of resin in deionized water, pouringthe slurry into a 1.8 cm. diameter glass column and allowing the resinto settle out and the excess water to drain off. A 0.1 percent w./v. eggalbumin solution was then run carefully onto the damp bed of resin andthe absorbance of the solution emerging from the bottom of the columnwas measured at a wavelength of 280 m using an Optica Densitronicspectrophotometer fitted with a 1 cm. path flow cell. The presence ofprotein in the solution was indicated by absorbance at this wavelengthwhich is believed to be due to the presence of tyrosine residues in theprotein. On the other hand, the absence of protein in the solutionleaving the column was indicated by low U.V. absorbance. As a furthertest for the presence of protein, fractions from the bottom of thecolumn were tested with phospho-tungstic acid. The presence of proteinwas indicated by a white precipitate. There was no precipitate formed onaddition of phosphotungstic acid when there was no protein present. Thistest with phosphotungstic acid affords a sensitive qualitative test forprotein in the range of 20 to 1000 ppm.

The resins were then regenerated and the presence or absence of proteinin the regenerant eluate (as indicated by the U.V. adsorbances and thephosphotungstic acid test) showed whether the resin had in fact absorbedprotein from the solution or not.

The following resins were tested:

A. Permutit Zeocarb 216 resin This resin is a phenol-formaldehydecondensate in granular form and contains OH and COOI-I groups. Itbehaves as a weakly acidic cation exchanger and corresponds to Erlichstype C (U.S. Pat. 2,446,913, Column 5, line 59 to 61). The column bedtested contained 35 g. (dry weight of resin, mesh size 14 52 (BritishStandard 410/62). The resin column was regenerated with 0.5N-HC1, at aflow rate of 5 ml./min. for 30 minutes and then washed with deionizedwater until the effluent was neutral. The resin column was kept coveredwith liquid throughout the experiment.

Upon running the test solution through the column at a flow rate of 5ml./min., after approximately 1 bed volume of albumin solution hadpassed into the column (thereby essentially displacing the deionizedwater which was on the column before the albumin solution was applied,i.e. the displacement volume), the effluent gave a precipitate withphosphotungstic acid and the recorder showed an abrupt rise in theabsorbance at 280 m from zero to a value essentially equal to that ofthe test solution. All fractions subsequently tested showed the presenceof protein. This showed that a large proportion of the protein was notbeing absorbed from the solution.

After rinsing the resin bed with water, the bed was regenerated withlN-HCl solution and 20 ml. fractions were again collected and testedwith phosphotungstic acid solution. No material giving a positive testwith phosphotungstic acid was eluted from the column showing that theresin had not taken up any significant amount of protein from the testsolution.

8 B. Amberlite IR-4B resin This is a weakly basic anion exchange resinof the synthetic amine-formaldehyde type (Erlichs type Asee U.S. Pat.No. 2,446,913, column 5 line 63). Again 35 g. dry weight of resin wasused. This was previously treated with 250 ml. of 0.5N-NaOH overnight toremove at least most of the soluble coloured material present in theresin and then washed extensively with water until neutral. 400 ml. ofthe test solution of albumin was passed into the column at a flow rateof 5 mls/min. Immediately after the displacement volume of water hademerged from the column the base line showed an abrupt rise of theoptical density at 280 m Simultaneous tests of 20 ml. fractions of theeffluent with phosphotungstic acid solution gave a white precipitateafter the first two fractions. This indicates that the protein waspassing through the resin with no significant uptake of protein by theresin.

The column was then rinsed with water and alkaline brine (5% NaCl+1%NaOH) was passed into the column. A quantity of coloured material waseluted from the column by the regenerant solution. However tests of theeffluent with phosphotungstic acid solution gave no significantturbidity or precipitate, thus indicating absence of protein. Thesetests clearly indicate that the synthetic amine-formaldehyde resin wasincapable of extracting protein from aqueous solution.

C. Epichlorhydrin cross-linked regenerated cellulose containingdiethylaminoethyl groups This resin was prepared by the method describedin Example 2 of U.S. Pat. 3,573,277, except that the regeneratedcellulose used has a mesh size of 30 to 50 mesh (British Standard410/62). 12 g. (dry weight) of the resin was used to form a 60 ml. bedvolume column. The bed was regenerated with 0.5 NaOH solution at a flowrate of 5 ml./min. for 30 minutes and washed with water until theeffluent was neutral. 400 ml. of the test solution of egg albumin wasthen passed through the bed at a flow rate of 5 ml./min. 20 fractions ofthe efiluent were collected and tested with phosphotungstic acidsolution as before. No precipitate was formed and no change inabsorbance at 280 m was noted as the solution was run through thecolumn. This indicates that the resin was taking up essentially all theprotein from the solution.

Upon regeneration with alkaline brine (5% brine NaCl +1% NaOH) 20 ml.fractions of the eluate were tested with phosphotun-gstic acid solutionand it was found that very heavy precipitates were formed in the thirdto the fifth fractions, after which the amount of precipitate decreased.The amount of the precipitate from each of the third to the fifthfractions was very heavy compared with the precipitate obtained from a20 ml. sample of the test albumin solution. The obsorbance at 280 mshowed a corresponding sharp peak corresponding to the third to fifthfractions.

This test showed that a cross-linked regenerated cellulose containinggroups capable of anion exchange can absorb protein from solution andthat the absorbed protein can be quickly and efliciently desorbed duringregeneration, the protein being eluted as a narrow zone of concentratedsolution.

Similar results to those found for Resin C were obtained with thefollowing Resins D and E.

D. Epichlorhydrin cross-linked regenerated cellulose in granular formcontaining carboxyl groups This resin which contains carboxymethylgroups was prepared by method described in Example 3 of U.S. Pat.3,573,277. It was regenerated by washing with acid. The successful useof this resin in the above test shows that a cross-linked regeneratedcellulose containing groups capable of cation exchange can be used toabsorb protein from solution in a readily desorbable form.

9. E. Formaldehyde cross-linked regenerated cellulose containingdiethylaminoethyl groups This resin was prepared by the method describedin Example 4 of U.S. Pat. 3,573,277.

The following resins were also tested:

F. Imac A17P resin This is a conventional synthetic ion exchange resinof the weak base type produced by the condensation of epichlorhydrinwith polyamines.

G. Amberlite IRA-401$ resin (chloride form) This is a porous strong baseanion exchanger in bead form derived from polystyrene crosslinked withdivinylbenzene.

H. Amberlite IRA-401.8 resin (hydroxide form) Using the above resins Fto H similar results were obtained to those obtained for resins A and B,i.e. it was found that the resins were incapable of taking up proteinfrom the test solution.

The invention will now be further described by way of example withreference to the accompanying drawings in which,

FIG. 1 is a block diagram of apparatus for use in treating effluentaccording to the invention,

FIG. 2 is a diagram of an experimental filter bed complex using aparticulate filter according to the invention,

FIG. 3 shows a simple experimental regeneration cycle,

FIGS. 4, 5 and 6 show steps in an alternative merrygo-round regenerationsystem, and

FIG. 7 shows a vertical cross section through a plant using the processof the invention.

Referring to the drawings, a receiving vessel, 1, is provided, adaptedto receive waste eflluents. For example, in a freezing works theefiluents may be from a slaughter board drain 2, a casing drain 3, askin wash drain 4 and/ or a paunch washings drain 5. However, it is tobe understood that in practice, it may be necessary or desirable totreat departmental wastes separately. From the receiving vessel 1, thewashings or efiiuent are passed through a mechanical pre-treatmentsection 6. In this pre-treatment section the treatment can consist ofpassing the material through a 60 mesh sieve, the sieve being providedwith suitable means whereby the collected material may be removed,either continuously or from time to time. Alternatively, on a largerscale a rotary vacuum filter can be used. Following mechanicaltreatment, the effiuent passes to a filter bed 7 in which the filter bedcomprises a granular or particulate resin material consisting of acrosslinked regenerated cellulose matrix into which have been introducedion exchange groups capable of anion exchange or of cation exchange.Passage of the efiiuent or washings through the bed 7 removesessentially all the protein therefrom. After such treatment, I havefound it preferable that the effluent be further treated by a fibrousresin in a scavenger bed 8. Following this, the effiuent may pass eitherdirectly through conduit 9, to a Waste discharge station, or may bepassed through a percolating filter or bone char filter 10 whereupon theoutgoing effluent from conduit 11 leading from filter 10 may bechorinated and re-used.

Both the filter bed 7 and scavenger bed 8 may be backwashed when desiredto dislodge solid matter from the resins. The backwash waters may bealso conducted to the filter 10 by means of conduits 12, 13, and 14.During the regeneration of the resin in the filter bed 7, the efiluentcontaining the released protein may be collected through conduit 15.Similarly, during regeneration of the fibrous resin in the scavenger bed8, released protein may be collected through conduit 17. Waste liquorswhich do not need to go through the filter 10 may be withdrawn from thefilter bed 7 and scavenger bed 8 through conduits 16 and 9 respectively.

Referring now to FIG. 2, the equipment shown in the block diagram ofFIG. 1 under reference 7, consists of a series of tanks 18 to 25, thefirst group 18-21 being connected in series, and the second group 22-25being also connected in series, the first group supplying an output 26,the second group supplying an output at 27, and both groups leading tobeds of fibrous resin. The two groups of tanks 18-21 and 22-25 arefilled with a cross-linked regenerated cellulose ion exchange materialand are supplied from a supply tank 28, which may be the pre-treatmenttank 6 shown in FIG. 1 (or may be an alternative tank), through leads29, 30 and 31. Although each of the tanks 18-25 may contain the sameresin, it is often desirable that one or more of the tanks in each setshould contain a resin capable of cation exchange whilst others containa. resin capable of anion exchange. The tanks may be of any suitablesize, for example, for experimental purposes, these tanks have been madein four inch diameter glass columns, and the settled heights of resinhave been about 12 inches high in each tank. The fiow rate, in thiscase, is approximately eight gallons per hour, and the dead volume ineach tank, about one gallon per tank. For a pilot plant, the tanks canbe approximately three feet in diameter, and ten feet high, holding atotal quantity of approximately one ton of resin. A plant having aninput of 1 million gallons per day would require a bed approximatelythirty five feet in diameter, and one to two feet deep. Of course, thearea of the bed could be spread over several tanks. These figures arebased on the ion exchangers described in US. Pat. 3,573,277, using thevery simplest form of operation.

In FIG. 3, a simple regeneration system is shown, in which the bedsshown diagrammatically at 32, comprise beds of granular cross-linkedregenerated cellulose ion exchange material in acid form, the beds 33granular beds in basic form, and beds 34 comprise further beds in whichthere is a resin in fibrous or sponge form, these forming a scaveningfilter. The fibrous form of resin may be, for example, diethylaminoethylcellulose (DEAE-cellulose), or carboxymethyl cellulose, which areparticularly useful for removing protein and fat which may break throughthe particulate resin beds as they approach complete exhaustion. Itshould be pointed out that it is not usually practicable to apply theraw effluent directly to the scavening filter, in view of the largeamounts of colloidal or suspended matter which may be present in the raweffiuent, and which would result in rapid clogging of the scavengingfilter. It is to be understood that when the beds 32 to 34 are in use,the beds 35, 36 and 37 (corresponding with the beds 32 to 34), are beingregenerated. Regeneration is effected by treating the ion exchangematerial, if the resin contains groups capable of cation exchange, with(e.g. hydrochloric acid) or a mixture of an acid and a mineral salt or,if the resin contains groups capable of anion exchange, with a solutionof alkali (e.g. NaOH) or a mixture of alkali and a mineral salt (e.g.NaCl). During the regeneration process, the protein is removed from thebeds as a relatively concentrated solution. When regeneration iscomplete, i.e. when the application of a further amount of regenerantsolution produces no significant quantities of protein, the bed iswashed with water to remove excess regcnerant solution, and the bed isthen ready for a further cycle of eifiuent purification. The processconsists generally, of alternating cycles of eflluent penetratration andregeneration of the resins bed. The period of time during which effluentis passed through a bed or series of beds before regeneration isstarted, depends on the purpose of the treatment of the efiluent. If thepurpose of the treatment is to achieve the maximum uptake of proteinfrom the effluent, then for a given weight of resin, it is necessarythat the capacity of the resin should be as nearly as possiblecompletely exhausted. Under these conditions, protein will leak or breakthrough the bed or beds in increasing amounts as the bed or beds becomemore and more nearly exhausted, so that a proportion of the resultantefliuent will still contain considerable proportions of protein. On theother hand, if the main purpose is to purify the efiiuent, then passingof effluent through the beds will be discontinued sooner and the extentto which the capacity is exhausted will be decided on by economic andother factors since, of course, it is not possible to completely freethe effluent of protein under normal commercial working.

I have found that, in certain extreme cases, even after the use of afibrous scavenger, there is some protein left in the efliuent, and inaddition, there is some odour due possibly to amines resulting fromdegradation of the protein. These may be reduced by passing the effluentin a final step through a percolating filter or bone char, activatedcarbon or coke filter (such as the filter referred to in connection withFIG. 1).

In relation to regeneration, a merrygoround regeneration cycle may beused as shown in FIGS. 4 and In this arrangement, three tanks 38, 39 and40 are provided which contain a particulate or granular form (e.g.

50-100 mesh) of cross-linked regenerated cellulose ion exchangematerial, and two sets of filters 41 and 42 contain a fibrous form ofresin such as DEAE-cellulose. In FIG. 4, the input 43 is led into tank38, the efliuent therefrom passing to tank 39, and then through thefibrous filter 41 to the output 44. In the meantime, the resin in tank40 and the fibrous filter 42 are being regenerated. In FIG. 5, the tank39 receives its input from conduit 45, the effluent is passed togranular filter set 40 then to fibrous filter set 42 to the output 46.In the meantime, granular filters 38 and fibrous filters 41 areregenerating. In FIG. 6, in the third step of the merrygoround cycle,the tank 40 receives input from the conduit 47, the etfluent thenpassing to tank 38, and the effluent then passing to fibrous filter 41;tank 39 and fibrous filter 42 are regenerating during this part of thecycle. This arrangement has the advantage that a newly regeneratedfilter bed is the second in a series of two filter beds, the first inthe set being a filter bed which has previously been used forpurification, and accordingly, its take up capacity is utilised to thebest advantage.

The water from which the protein and fat has been removed, may also bepassed to waste, but usually this water will have been purifiedefficiently, to enable the water to be reused for certain roughcleansing purposes, for example, hide washing.

In FIG. 7 there is shown semi-diagrammatically a plant for operating theprocess of the invention. This consists of two tanks 100 eachconstructed from stainless steel and each being about four feet indiameter and about six feet in height. Their domed bottoms are connectedto outlet pipes 101 leading a manifold 102 having three outlet valves103, 104 and 105. A layer of gravel A in. screened) 106 some 9 ins. indepth was placed in the bottom of each tan-k 100. A spiral distributorpipe 107 having a side outlet 108 was positioned on top of layer 106 andthis in turn was covered with a 3 in. deep layer 109 of stone chippingsAs" screened).

The tanks 100 were partly filled with water and sufficient resin wasthen added with stirring so that on settling the resin formed beds 110some 12 ins. in depth.

Some four feet above the top of the bed of resin were positioned in eachtank spray rings 111 through which liquid can be introduced into thetank.

Reference numeral 112 represents a source of effluent to be treated, 113a source of deionized water and 144 a source of regenerant solution. Bymeans of suit-able pipework and suitable manipulation of valves 115 to119 any of the three liquids, i.e. effluent, deionized water orregenerant, can be introduced at will into the tanks 110.

The beds of resin can be back-washed when desired by pumping waterthrough pipes 108 and the spiral distributor pipes 107, the backwashingsbeing removed 12 from the tank through overflow pipes 120. Pipes 120 areconnected to an overflow collar 121, the overflow of solids beingprevented by a mesh screen 122.

The following Examples serve to illustrate the invention further.

EXAMPLE 1 Using the apparatus of FIG. 7, a bed 110 of 50 to mesh(British Standard 410/62) epichlorhydrin cross-linked regeneratedcellulose containing diethy aminoethyl groups in the free base form wasformed in each of the tanks 100. This ion exchange resin was made inaccordance with the directions of Example 2 of US. Pat. 3,573,277, butusing viscose of 50 to 100 mesh size (British Standard 410/62).

Meat works efiiuent, which had been pretreated by submission to airflotation and settling to remove fat and insoluble protein, was passedinto the tanks 100 through the spray rings 111 from the tank 112. Thisefliuent resulted from slaughter of sheep and cattle and had a pH of6.5. The efiiuent was passed from the tank 112 through spray rings 111and the plant was run with the tanks 100 completely full of liquid. Byadjustment of the outlet valves 103 to the flow of effluent through thebeds was maintained at approximately 1 gallon per square foot per minutegiving a total flow of approximately 24 gallons per minute for the twotanks together. In this way, during a working cycle of 7 hours, a totalof about 10,000 gallons of effluent was treated.

The pretreated efiluent was brown in colour with a C.O.D. (ChemicalOxygen Demand) value which varied between about 500 and 2000. TheChemical Oxygen Demand (C.O.D.), is the quantity of oxygen, expressed inppm, consumed under specific conditions, in the oxidation of the organicand oxidizable inorganics contained in water and waste water, correctedfor influence of chlorides. For a description of its estimation seeMethods for Examination of Water and Waste Water, twelfth edition,Public Health Association, New York, N.Y. 1965 (pages 510'514).

The eluate from the ion exchange beds was water clear during most of theworking cycle with a faint yellow colour appearing towards the end ofthe working cycle. Continuous measurement of the COD. value of theeluate showed an average reduction of 90% compared With the pretreatedefliuent applied to the beds of resin. During most of the cycle,breakthrough of protein (as indicated by the phosphotungstic acidprecipitation test) was absent, except towards the end of the cycle whentrace amounts appeared in the efiiuent.

At the end of the 7 hour cycle the beds were regenerated by applying 1bed volume of alkaline brine regenerant (1% NaOH 3.5% NaCl) from thetank 114 to each ion exchange bed 110 after allowing the effluent levelto drain down to the surface of the resin. The alkaline brine was runslowly into the beds 110 and allowed to remain for 1 hour, after whichit was slowly displaced by adding water on top of the beds 110. Most ofthe protein taken up by the resin was displaced in little over 1 bedvolume of liquid.

The regenerant solution coming from the beds was almost black in colourand the pH of the solution was about 11. The pH of the regenerantsolution was then adjusted to about 5 byadding sulphuric acid, uponwhich a copious fine precipitate of protein formed.

Steam was then blown through the solution to raise the temperature to 80C. to cause coagulation of the protein into lumps. The co-agulatedprotein was then separated from the mother liquor on an 80 mesh (BritishStandard 410/62) wire basket strainer. The mother liquor was almost freeof protein and was colourless. After draining for some hours the proteincake had a solids content of about 10% and was dried in a hot airtunnel.

After regeneration was completed the resin beds were.

washed with about three bed volumes of water and effluent was thenapplied for the next 7 hour working cycle.

to lbs. of protein were recovered from each bed per cycle on theaverageand virtually no protein was lost in the final efiluent or themother liquor from the regenerant after coagulating the protein. Therecovered protein was used as poultry food.

Occasionally (for example every 10 or 20 cycles) the beds were freedfrom insoluble matter by backwashing with water through the pipe 108 andspiral distribution pipe 107 the backwashings being swept out of thetank through pipe 120 and discarded.

The resin can be used many times and it has been found that the resinused in this Example shows no dim inution in activity even after severalhundred cycles.

The following Table shows the amino acid composition of two samples ofprotein recovered from the pilot plant of FIG. 7. For comparison thecorresponding compositions of some related proteins are also listed.

TABLE 1 [Grams amino acid/l6 grams nitrogen] mately one bed volume(about 600 ml.) of regenerant (3% by weight NaCl, 1% by weight NaOH).The regenerant eluate was then acidified to pH 4 and sodiumhexametaphosphate (5 g.) added. The precipitate of protein wascentrifuged off and dried to give approximately g. of protein. Theregenerated bed was washed and the cycle repeated.

The factory had a throughput of about 20,000 birds daily which werekilled, plucked and dressed. A proportion of the throughput was cookedin pressure cookers to provide chicken meat for chicken pies. Theefiiuent contained not only the effluent from the killing, plucking anddressing section but also the cooking liquors.

EXAMPLE 3 The effluent from a natural rubber latex processing plant,which contains the liquor resulting after coagulation of the rubber fromthe latex, is a dark liquid which can be shown to contain protein.

Recovered solids Blood proteins (reference below) Fraction Hemo- SerumAmino acid A B Fibrin globins proteins Casein Lysine 8. 8 8.5 9. 1 9. 110. 0 8. 5 Histidine 3. ,9 5. 9 2. 9 8.0 3. 3 3. 2 Arginine 4. 4 4. 77.8 3. 9 5. 8 4. 2 Aspartic acid. 14. 3 0. 4 11. 9 9. 8 10. 3 7. 0Threonine 7. 0 4. 5 7. 3 5-6 12. 6 4. 5 Serine 2 7. 7 5. 7 12. 5 5. 518. 2 6. 8 Glutamic acid 19. 3 10. 0 15.0 8. 1 14. 2 23.0 6. 6 3. 2 5. 34. 7 5. 5 13. 1 5.5 3.5 5.4 5.3 2.0 2.1 8.8 6.8 4.0 9.8 3.3 Trace Trace3. 8 1. 0-2. 2 7. 0 O. 8 11.0 8.2 5.6 9.0 7.5 7.7 2. 8 3. 2 2. 6 1-3 4.03. 5 Isoleueine 5. 5 4. 1 5.6 0-2 3. 4 7. 5 Leucine 17. 1 15. 0 7. 1 14.4 10. 1 10. 0 Tyrosine 1 5. 5 3. 2 6.0 2. 9 5. 5 6, 4 Phenylalanine. 9.9 7.9 4. 5 7-8 5. 2 6. 3 Ammonia 1. 4 1. 1

Total 140 105 1 Multiplied by 1.1 to allow (roughly) for decompositionin hydrolysis. 2 Multiplied by 1.4, same reason.

No'rE.-Reference for comparative analyses: The Amino Acid Composition ofProteins syn; goods R. J. Block and D. Bolling (1951, 2d Ed.) Publishedby: Charles C. Thomas,

Both samples A and B contain excellent protein from a nutritional pointof view as shown by the amino acid composition. From a nitrogen analysisit was estimated that sample A contained about 61.3% protein whereassample B contained about 73.9% protein.

EXAMPLE 2 This Example illustrates the use of a dichlorhydrincross-linked regenerated cellulose resin containing carboxymethyl groupsfor purifying an efiluent from a poultry processing factory. The resincan be prepared by the procedure of Example 3 of U8. Pat. No. 3,573,277,except that the 5 ml. of epichlorhydrin is replaced by 5 ml.dichlorhydrin.

The resin was slurried and poured in a conventional manner into achromatography column 5 cm. in diameter so as to form a columnapproximately 30 cm. long.

The efiiuent from the poultry processing factory was filtered by passagethrough an 80-mesh screen (British Standard 410/62) to remove suspendedsolids and then subjected to air flotation to remove at least part ofthe protein in the efliuent. After scraping off the scum formed by theair flotation treatment the efliuent was ready for passage through thebed (30 cm. x 5 cm.) of ion exchange resin.

5 gallons of the thus-treated efiiuent were passed through the bed ofresin. Monitoring of the U.V. absorbance of the eluate at 280 mu showedthat the majority of the remaining protein was being asborbed by theresin. Regeneration was accomplished using approxi- The procedure ofExample 2 was repeated except that the resin used was Resin C, i.e.epichlorhydrin crosslinked regenerated cellulose containing DEAE groups.The eluate was water clear and was shown by monitoring its U.V.absorbance at 280 me to be essentially protein free. The exhausted bedof resin could be regenerated as described in Example 2.

EXAMPLE 4 EXAMPLE 5 Example 4 is repeated except that Resin E is used inplace of Resin C with equally good results.

EXAMPLE 6 The procedure of Example 4 is followed but the eluate from thebed of Resin C is then passed through a bed of fibrous DEAE-cellulose.

15 EXAMPLE 7 The procedure of Example 4 is repeated with the addition ofthe final step of passing the resulting eluate through a bone charfilter.

What is claimed is:

1. A process for the purification of a waste effluent containing proteinwhich comprises the steps of:

(a) passing said effluent through a bed of a particulate cellulose-basedion exchange material so as to adsorb protein from said effluent, saidion exchange material comprising a matrix of cellulose chains inregenerated form which carry ion exchange groups and which arecross-linked by residues of at least one cross-linking agent which isitself free from ion exchange groups, the degree of cross-linking insaid ion exchange material being from 1 to 10% by weight calculated asthe weight of reacted crosslinking agent based on the weight ofcellulose; and

(b) subsequently regenerating said ion exchange material for use in afurther cycle.

2. A process for the purification of an efiluent containing proteinwhich comprises the steps of:

(a) submitting the efiluent to a pretreatment so as to effectprecipitation of coagulable proteins and at least a substantial part ofany fat present;

(b) separating the precipitated solids from the effluent;

(c) passing the effluent through one or more beds of a granular ionexchange material so as to adsorb protein from the efiluent, which ionexchange material comprises a matrix of cellulose chains in regeneratedform which carry ion exchange groups linked thereto and which arecross-linked by the residues of a crosslinking agent which is itselffree from ion exchange groups, the degree of cross-linking being from'lto 10% by weight calculated as the amount of crosslinking agent that hasreacted with a given weight of cellulose;

(d) recovering from the bed or bed of the ion exchange material anessentially protein-free liquid;

and (e) regenerating thebed or beds of the ion exchange material.

References Cited UNITED STATES PATENTS 2,446,913 8/1948 Erlich 2601 12 R3,697,419 7/1969 Grant 210-27 3,314,880 4/1967 Rubin 21044 3,409,60511/1968 Florini 2601 12 R 2,992,215 7/1961 Bullock et a1. 21037-X3,122,456 2/1964 Meier et a1. 2 21037 X 3,446,794 5/1969 Knight et a1.210-24 X 3,234,199 2/1966 Reid 260-112R 3,487,064 12/1969 Swanson eta1.- 260112 R 3,573,277 3/1971 Grant 260231 OTHER REFERENCES Petersonand Sober, JACS, Feb. 20, 1956, pp. 751-755.

JOHN ADEE, Primary Examiner IVARS CINTINS, Assistant Examiner US. Cl.X.R.

